We and others have recently shown that alpha3Beta1, alpha4Beta1, and alpha6Beta1 directly associate with Transmembrane 4 Superfamily (TM4SF) proteins CD9, CD53, CD63, CD81, and CD82. Also, we found that integrins and TM4SF proteins co-localize at the cell surface, in cathepsin D positive granules, and in cell lamellipodia, but not in focal adhesion complexes. Notably, treatment with soluble anti-CD82 mAb triggered the localization of P-tyr-containing proteins into focal adhesion complexes, suggesting that TM4SF proteins may be dynamic regulators of integrin function. Like integrins, TM4SF proteins can regulate cell movement, growth, spreading, and associated signaling pathways. We hypothesize that physical association between integrins and TM4SF proteins accounts for their overlapping functional attributes. First, we will determine the influence of TM4SF proteins on integrin adhesion, ligand binding, tethering and adhesion strengthening under shear flow, and cell surface clustering. Second, specific sites required for integrin/TM4SF interaction will be identified. Mutant integrins will be generated that lack TM4 interaction sites, and mutant TM4SF proteins will be made that lack TM4/integrin and/or TM4/TM4 interaction sites. Third, we will examine integrin/TM4SF association with respect to cell morphology and signaling, with particular emphasis on the roles of rac, rho, and cdc42, and TM4SF-associated phosphatase activity. Fourth, we will test the hypothesis that TM4 associations may be critical for alpha3 and alpha4 integrin-dependent control of cell migration and growth. The proposed studies will utilize i) our many mAb's and cDNA's for integrins and TM4SF proteins, ii) a novel technical approach for removing integrin surface expression, and iii) cells available from alpha3 and alpha4 "knockout" mice. Results showing that integrin/TM4 associations regulate cell morphology, migration, and proliferation will be highly relevant towards understanding of tumor cell growth and metastasis in vivo.